Darryl D. Glubrecht

The Absence of Human Equilibrative Nucleoside Transporter 1 Is Associated with Reduced Survival in Patients With Gemcitabine- Treated Pancreas Adenocarcinoma

Purpose: Gemcitabine monotherapy is the standard palliative chemotherapy for pancreatic adenocarcinoma. Gemcitabine requires plasma membrane nucleoside transporter proteins to efficiently enter cells and exert it cytotoxicity. In vitro studies have demonstrated that deficiency of human equilibrative nucleoside transporter 1 (hENT1), the most widely abundant and distributed nucleoside transporter in human cells, confers resistance to gemcitabine toxicity, but the distribution and abundance of nucleoside transporters in normal and malignant pancreatic tissue is unknown. Experimental Design: We studied tumor blocks from normal pancreas and 21 Alberta patients with gemcitabine-treated pancreatic cancer. Immunohistochemistry on the formalin-fixed, paraffin-embedded tissues was performed with specific hENT1 and human Concentrative Nucleoside Transporter 3 monoclonal antibodies and scored by a pathologist blinded to clinical outcomes. Results: hENT1 was detected in normal Langerhan cells and lymphocytes but not in normal glandular elements. Patients in whom all adenocarcinoma cells had detectable hENT1 had significantly longer median survivals from gemcitabine initiation than those for whom hENT1 was absent in a proportion (10 to 100%) of adenocarcinoma cells (median survival, 13 versus 4 months, P = 0.01). Immunohistochemistry for human Concentrative Nucleoside Transporter 3 revealed moderate to high-intensity staining in all adenocarcinoma tissue samples. Conclusions: Patients with pancreatic adenocarcinoma with uniformly detectable hENT1 immunostaining have a significantly longer survival after gemcitabine chemotherapy than tumors without detectable hENT1. Immunohistochemistry for hENT1 shows promise as a molecular predictive assay to appropriately select patients for palliative gemcitabine chemotherapy but requires formal validation in prospective, randomized trials.

DEAD box 1: a novel and independent prognostic marker for early recurrence in breast cancer

Breast cancer is a heterogeneous disease characterized by diverse molecular signatures and a variable response to therapy. Clinical management of breast cancer is guided by the expression of estrogen and progesterone receptors and HER2 amplification. New prognostic and predictive markers, as well as additional targets for therapy, are needed for more effective management of this disease. Gene expression microarrays were probed with RNAs from 176 primary breast cancer samples and tissue microarrays immunostained with anti-DDX1 antibody, an antibody to DEAD box protein DDX1, a putative RNA-RNA and RNA-DNA unwinding protein normally found in the nucleus. Half of the patient cohort had experienced early relapse despite standard adjuvant therapy, but were otherwise matched for estrogen receptor and HER2 status, stage and duration of follow-up. Here, we identify DDX1 RNA overexpression as an independent prognostic marker for early recurrence in primary breast cancer, with a hazard ratio of 4.31 based on logrank analysis of Kaplan–Meier curves. Elevated levels of DDX1 protein in the cytoplasm also independently correlate with early recurrence with a hazard ratio of 1.90. In conclusion, our data indicate a strong and independent association between poor prognosis and deregulation of the DEAD box protein DDX1. We propose that elevated levels of DDX1 RNA or the presence of DDX1 in the cytoplasm could serve as an effective prognostic biomarker for early recurrence in primary breast cancer.

Nuclear Factor I Regulates Brain Fatty Acid-Binding Protein and Glial Fibrillary Acidic Protein Gene Expression in Malignant Glioma Cell Lines

Glial fibrillary acidic protein (GFAP), an intermediate filament protein normally found in astrocytes, and the radial glial marker brain fatty acid-binding protein (B-FABP; also known as FABP7) are co-expressed in malignant glioma cell lines and tumors. Nuclear factor I (NFI) recognition sites have been identified in the B-FABP and GFAP promoters, and transcription of both genes is believed to be regulated by NFI. Here, we study the role of the different members of the NFI family in regulating endogenous and ectopic B-FABP and GFAP gene transcription in human malignant glioma cells. We show by gel shifts that all four members of the NFI family (NFIA, NFIB, NFIC, and NFIX) bind to B-FABP and GFAP NFI consensus sites. Over-expression of NFIs, in conjunction with mutation analysis of NFI consensus sites using a reporter gene assay, supports a role for all four NFIs in the regulation of the GFAP and B-FABP genes. Knock-down of single or combined NFIs reveals promoter-dependent and promoter-context-dependent interaction patterns and suggests cross talk between the different members of the NFI family. Our data indicate that the NFI family of transcription factors plays a key role in the regulation of both the B-FABP and GFAP genes in malignant glioma cells.

A fatty acid-binding protein 7/RXRβ pathway enhances survival and proliferation in triple-negative breast cancer.

FABP7 has been implicated in tumour cell proliferation, cell migration, and poor prognosis in patients with high‐grade astrocytoma and melanoma. In this study, we examine FABP7 expression in a cohort of 176 primary breast cancers by gene profiling and tissue microarray immunostaining. We show that FABP7 is significantly up‐regulated in triple‐negative breast cancer. Elevated FABP7 levels are associated with poor prognosis, absence of oestrogen and progesterone hormone receptors (ER, PR) and HER2, increased cell proliferation, and high tumour grade. Depletion of FABP7 in the ER/PR‐negative cell line, MDA‐MB‐435S, significantly reduced cell growth rate and sensitized the cells to growth inhibition by omega‐3 docosahexaenoic acid (DHA). A target of DHA‐bound FABP7 in the nucleus is RXRβ, a retinoid‐activated nuclear receptor that functions as a transcription factor by either homodimerizing or heterodimerizing with other nuclear receptors such as PPARs. Based on our microarray data, RXRβ, like FABP7, is an adverse prognostic factor for breast cancer. We propose that the DHA‐FABP7‐RXRβ pathway promotes cell survival/proliferation in triple‐negative breast cancer. Targeting this pathway may thus provide an alternate route for the treatment of triple‐negative breast cancer. Copyright

The early isoform of disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina.

ABSTRACT The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.

Splice-mediated motif switching regulates disabled-1 phosphorylation and SH2 domain interactions.

ABSTRACT Disabled-1 (Dab1) plays a key role in reelin-mediated neuronal migration during brain development. Tyrosine phosphorylation of Dab1 at two YQXI and two YXVP motifs recruits multiple SH2 domains, resulting in activation of a wide range of signaling cascades. However, the molecular mechanisms underlying the coordinated regulation of Dab1 downstream effectors remain poorly understood. Here, we show that alternative splicing results in inclusion of different combinations of YQXI and YXVP motifs in Dab1 isoforms during development. Dab1 variants with partial or complete loss of YQXI motifs are preferentially expressed at early developmental stages, whereas the commonly studied Dab1 is predominantly expressed at late developmental stages. Expression of Dab1 variants in 293T and Neuro2a cells reveals reduced levels or absence of tyrosine phosphorylation in variants that have lost one or both YQXI motifs. We further demonstrate that Dab1 variants differ in their abilities to activate Src and recruit distinct SH2 domains involved in specific downstream signaling pathways. We propose that coordinated expression of specific Dab1 isoforms in different populations of cells in the developing brain contributes to precise neuronal migration by modulating the activity of subsets of Dab1 downstream effectors.

Expression of AP‐2δ in the developing chick retina

AP‐2 is a family of transcription factors that play important roles during embryonic development. Two AP‐2 genes, AP‐2α and AP‐2β, have previously been characterized in chick retina. Here, we demonstrate that a third member of the chicken AP‐2 family, AP‐2δ, is primarily expressed in the retina and brain, with highest levels at embryonic days 7 to 11. By in situ hybridization and immunohistochemical analysis, we show that AP‐2δ RNA and protein are found in a subset of ganglion cells in embryonic chick retina. Co‐immunostaining with anti‐Brn3a and anti–AP‐2δ antibodies indicates that approximately one‐third of Brn3a‐positive ganglion cells express AP‐2δ. AP‐2δ RNA but not AP‐2δ protein is also found in cells located in the outer half of the inner nuclear layer. The spatial and temporal distribution of AP‐2δ protein in the retina suggests a transient role in a subset of late‐born ganglion cells likely involving axonal trafficking or pathfinding. Developmental Dynamics 237:3210–3221, 2008.

CRABP1 is associated with a poor prognosis in breast cancer: adding to the complexity of breast cancer cell response to retinoic acid

BackgroundClinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer.MethodsCRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis.ResultsCompared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.ConclusionsCRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

Calcineurin regulates Nuclear Factor I dephosphorylation and activity in malignant glioma cell lines

Background: Nuclear factor I (NFI) phosphorylation controls the expression of glial genes associated with migration. Results: NFI is dephosphorylated and activated by a cleaved form of calcineurin in malignant glioma cells. Conclusion: NFI transcriptional activity in malignant glioma is regulated in part by calcineurin. Significance: Regulation of NFI by calcineurin provides a novel approach to control the expression of genes associated with migration/infiltration in malignant glioma. Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 years. Nuclear factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP; FABP7) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcriptional activity, and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is up-regulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties.

Association between cytoplasmic CRABP2, altered retinoic acid signaling, and poor prognosis in glioblastoma.

Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid‐binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose‐dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA‐induced RAR activation. Levels of CRYAB, a small heat shock protein with anti‐apoptotic activity, and GFAP, an astrocyte‐specific intermediate filament protein, are greatly reduced in CRABP2‐depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti‐apoptotic pathway, thereby enhancing proliferation and preventing RA‐mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients. GLIA 2016;64:963–976